Computational protocol: A large-scale multiomics analysis of wheat stem solidness and the wheat stem sawfly feeding response, and syntenic associations in barley, Brachypodium, and rice

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Protocol publication

[…] Total RNA was extracted for three replicates of control and infested stem tissues from the two cultivars, as previously described (Cagirici et al. ). RNA sequencing (RNA-Seq) was performed on the isolated RNA samples with RNA integrity numbers of greater than 7. The library was prepared using a TruSeq RNA Library Preparation Kit (Illumina, San Diego, CA, USA), following the manufacturer’s instructions, and sequencing was performed using an Illumina Hi-Seq 2000 platform. The RNA-Seq reads were trimmed using Sickle (v.1.33) before being aligned to the T. aestivum chromosome 3B assembly using GMAP (v.2017-02-25; Wu and Watanabe ). The aligned reads were then compared to a GFF file containing annotated transcripts of T. aestivum (variety Chinese Spring) and the read counts were obtained using HTSeq-count (−m union; Anders et al. ). Raw read counts were filtered using the Noleaven R package (https://github.com/topherconley/noleaven) to eliminate those with zero or low counts across the entire dataset. The identification and functional annotation of differentially expressed genes (DEGs) between the control and WSS-treated stem samples of Choteau and Scholar were performed as previously described (Cagirici et al. ). […]

Pipeline specifications

Software tools GMAP, HTSeq
Application RNA-seq analysis
Organisms Cephus cinctus, Triticum aestivum, Hordeum vulgare, Oryza sativa, Caenorhabditis elegans, Drosophila melanogaster
Diseases Wheat Hypersensitivity
Chemicals Phosphates